The primitive linear heart tube (dorsal vessel) of the fruit fly Drosophila melanogaster provides a unique model system to study the genetic control mechanisms of the heart development. Using RNAi (RNA interference) in developing Drosophila embryos we performed genome-wide screening of regulatory genes that are involved in the Drosophila heart development. From this screen, both novel genes and known genes with mammalian homologues and with previously undescribed functions in heart development have been identified. Among candidate genes, we have further characterized the sprite (sprt) gene, which encodes a protein containing a PDZ domain. sprt is expressed in various tissues including the heart, gonadal mesodermal cells and malpighian tubules during embryogenesis. sprt is also expressed in a small group of cells undergoing tissue remodeling such as ectodermal cells in segmental grooves between somatic muscles. During cardiogenesis, cardial and pericardial cells start to express sprt at the stage 12, and its expression was maintained until maturation of the heart tube. To further characterize sprt function during cardiogenesis, we also generated both sprt mutant flies by P-element excision and transgenic fly lines expressing various Sprite proteins. We found that sprt mutation drives changes in cardial cell shape and consequently causes disorganized heart. Forced-expression of the truncated form of the Sprite lacking a PDZ domain also resulted in disorganized heart. In the heart tube, the Sprite protein showed a polarized localization distinct from alpha-Spectrin that predominantly localized to the lateral side of the cardial cells. To identify signaling molecules that may affect the Sprite function in cardiac morphogenesis, we selected several mutants, which, from our RNAi screen, showed a similar heart phenotype as the sprite mutant hearts and investigated localization of the Sprite protein in cardial cells by immunostaining and confocal microscopy. Among genes we tested, we found that the heart is disorganized in the faint sausage (fas) and walrus (wal) mutant embryos. Interestingly, we found that localization of the Sprite protein was disturbed in these embryos and detected the truncated Sprite protein by western blot analysis, which is probably generated by specific protease cleavage. Forced expression of the dominant-negative form of CDC42 caused mis-localization of the Sprite protein, resulting in disorganized hearts. Taken together, our results indicate that sprt plays a role in the late steps of cardiac morphogenesis and identify a potential signaling pathway that regulates the Sprite function during the maturation of the heart tube. Because the genetic control mechanisms of the embryonic cardiac development of the Drosophila are well conserved in mammalian heart development, identifying cardiogenic regulatory genes in Drosophila should help us understand the genetic control mechanism of the mammalian cardiac morphogenesis.